The present invention relates to a new protein called RB18A for xe2x80x9cRecognized By PAb1801 moAntibodyxe2x80x9d, which is a p53 regulatory protein, to the nucleotide sequence encoding said protein, and to the diagnostic and therapeutic applications thereof.
The p53 protein plays an important and complex role in cells. In normal cells, wild-type p53 is involved in genome stability (Lane, 1992) and in reparation of DNA lesions (Kastan et al., 1991; Kastan et al., 1992; Fritsche et al., 1993). In tumor cells, overexpression of wild-type p53 induces, depending on cell type. a G1 cell-cycle growth arrest (Ginsberg et al., 1991; Mercer et al., 1991) or apoptosis in vitro (Yonish-Rouache et al., 1991; Johnson et al., 1993) and in vivo (Shaw et al., 1992; Radinsky et al., 1994). These functions of p53 are related to its property to transactivate (Kastan et al., 1992; Scharer and Iggo, 1992; El-Deiry et al., 1993) or to repress transcription of different genes (Ginsberg et al., 1991; Santhanam et al., 1991; Subler et al., 1992) and to inhibit cellular DNA replication (Miller et al., 1995; Cox et al., 1995).
The regulatory functions of p53 are associated to specific domains (Soussi et al., 1990). Its N-terminal domain acts as a trans-activation domain (Fields and Jang, 1990; O""Rourke et al., 1990; Raycroft et al., 1990). Its central domain contains a sequence-specific DNA binding site (Wang et al., 1993; Pavietich et al., 1993; Bargonetti et al., 1993), which interacts with two copies of the 10 bp sequence 5xe2x80x2-PuPuPuC(A/T)(T/A)GPyPyPy-3xe2x80x2, separated by up to 13 bp (El-Deiry et al., 1992; Funk et al., 1992). This DNA element is present in promotor of several genes including WAF1 (El-Deiry et al., 1993), GADD45 (Kastan et al., 1992), the muscle creatine kinase gene (Weintraub et al., 1991), and the MDM2 gene (Barak et al., 1993). Other domains modulate the specific interaction of p53 with DNA. Indeed, a tetramerisation domain localized within the C-terminal stabilizes this interaction (El-Deiry et al., 1992; Funk et al., 1992; Arrowsmith and Morin, 1996). In addition, the p53 C-terminal domain contains a negative autoregulation site of sequence-specific DNA binding (Hupp et al., 1992). The C-terminal domain also carries a nonspecific DNA binding activity (Wang et al., 1993). This property has been associated to a DNA annealing and strand-transfer activities of p53 (Reed et al., 1995).
Thus, proteins which interact with one of these p53 domains should regulate p53 functions. Different proteins have been described as interacting with p53: viral SV40 large T antigen (Lane and Crawford, 1979), adenovirus ElB 55K (Sarnow et al., 1982), EBV BZLFI (Zhang et al., 1994), cellular Hsp70 (Pinhasi-Kimhi, 1986), MDM2 (Mommand et al., 1992), TBP (Seto et al., 1992), WT1 (Maheswaran et al., 1993), the Epstein-Barr virus/C3d receptor (CR2, CD21) (Barel et al., 1989) and more recently PACT (Simons et al., 1997). WO 95/14777 also discloses p53-binding polypeptides, called WBPI and p53UBC. Furthermore, monoclonal antibodies (moAb) directed against specific p53 domains are also p53 binding proteins which allow to analyze the role of p53 domains and to distinguish between wild-type or mutant p53 forms. PAb1620 moAb specifically recognized a conformational epitope only expressed on wild-type p53 (Milner et al., 1987), while PAb240 moAb recognized a sequencial epitope localized in the central region of p53 and demasked only on mutated p53 (Gannon et al., 1990). Other anti-p53 moAb as PAb1801 (binding to aminoacids 46 to 55 of p53) and DO1 (binding to aminoacids 21 to 25 of p53) or PAb421 (binding to aminoacids 371 to 380 of p53) were found to be directed against either the N or the C terminal domains of the p53 protein, respectively (Legros et al., 1994). PAb421 moAb activates the sequence-specific DNA binding activity of p53, in a similar manner to phosphorylation or binding of bacterial Hsp70(dnak) on the p53 C-terminal domain (Hupp et al., 1992).
The authors of the present invention have now identified a new cellular protein, with an apparent molecular weight of 205 kDa. This protein was called RB18A for xe2x80x9cRecognized By PAb1801 moAbxe2x80x9d, as isolated after immunological screening of a cDNA expression library using the specific anti-p53 moAb (monoclonal antibody), PAb1801. Although no significant homology has been found with p53 at the level of nucleotide or deduced-protein sequence, RB18A protein shared some antigenic epitopes with p53 as recognized by different anti-p53 moAbs. Furthermore, RB18A protein shares some identical functional properties with the p53 protein, i.e. DNA-binding, homo-oligomerization, binding to p53 and activation of the sequence specific DNA binding function of p53.
In the instant application,
SEQ ID no1 represents the cDNA sequence encoding the human RB18A protein.
SEQ ID no2 represents the aminoacid sequence of the human RB18A protein.
The fragments of the human RB18A protein of interest are the following:
the aminoacid fragment from aminoacid 436 to aminoacid 1566, encoded by the nucleotide fragment from nucleotide 1541 to nucleotide 4933;
the aminoacid fragment from aminoacid 436 to aminoacid 1228, encoded by the nucleotide fragment from nucleotide 1541 to nucleotide 3919;
the aminoacid fragment from aminoacid 436 to aminoacid 927, encoded by the nucleotide fragment from nucleotide 1541 to nucleotide 3014;
the aminoacid fragment from aminoacid 1537 to aminoacid 1566, encoded by the nucleotide fragment from nucleotide 4846 to nucleotide 4933;
the aminoacid fragment from aminoacid 1234 to aminoacid 1566, encoded by the nucleotide fragment from nucleotide 3935 to nucleotide 4933;
the aminoacid fragment from aminoacid 1234 to aminoacid 1406, encoded by the nucleotide fragment from nucleotide 3935 to nucleotide 4453, which represents the p53 binding and homo-oligomerization domains;
the aminoacid fragment from aminoacid 927 to aminoacid 1406, encoded by the nucleotide fragment from nucleotide 3014 to nucleotide 4453, which represents the DNA binding domain.
A subject of the present invention is thus an isolated nucleic acid comprising a sequence selected from the group consisting of a fragment from nucleotide 1541 to nucleotide 4933 of SEQ ID no1, a fragment from nucleotide 1541 to nucleotide 3919 of SEQ ID no1, a fragment from nucdeotide 1541 to nucleotide 3014 of SEQ ID no1, a fragment from nucleotide 4846 to nucleotide 4933 of SEQ ID no1, a fragment from nucleotide 3935 to nucleotide 4933 of SEQ ID no1, a fragment from nucleotide 3935 to nucleotide 4453 of SEQ ID no1, a fragment from nucleotide 3014 to nucleotide 4453 of SEQ ID no1, and a homologous nucleic acid sequence thereof.
More particularly the present invention relates to an isolated nucleic acid having a sequence selected from SEQ ID no1, and a homologous nucleic acid sequence thereof.
xe2x80x9cA homologous nucleotide sequencexe2x80x9d is understood as meaning a sequence which differs from the sequences to which it refers by mutation, insertion, deletion or substitution of one or more bases.
Preferably, such homologous sequences show at least 70% of homology, preferably 80% of homology, more preferably 90% of homology with SEQ ID no 1 or fragments thereof, as above defined.
A polynucleotide of the invention, having a homologous sequence, hybridizes to the sequences to which it refers, preferably under stringent conditions. Parameters that define the conditions of stringency depend upon the temperature at which 50% of annealed strands separate (Tm).
For sequences comprising more than 30 nucleotides, Tm is calculated as follows:
Tm=81.5+0.41(% G+C)+16.6 Log (positive ion concentration)xe2x88x920.63(% formamide)xe2x88x92(600/polynucleotide size in base pairs)(Sambrook et al, 1989).
For sequences comprising less than 30 nucleotides, Tm is calculated as follows:
Tm=4(G+C)+2(A+T).
Under appropriate stringent conditions avoiding the hybridization of non specific sequences, hybridization temperature is around from. 5xc2x0 C. to 30xc2x0 C., preferably from 5xc2x0 C. to 10xc2x0 C. below the calculated Tm, and hybridization buffer solutions that are used are preferably solutions with high ionic strength, such as an aqueous 6xc3x97SSC solution for example.
A nucleotide sequence homologous to SEQ ID no 1 means a nucleotide sequence which differs from SEQ ID no 1 by mutation, insertion, deletion or substitution of one or more bases, or by the degeneracy of the genetic code so long as it codes for a polypeptide having the biological activity of RB18A protein, as defined below.
Said homologous sequences include mammalian genes coding for the RB18A protein, preferably of primate, cattle, sheep, swine, or rodent, as well as allelic variants.
Polynucleotides of the invention are useful as probes for nucleic acid hybridization to detect the transcription rate and abundance of RB18A mRNA in individual lymphocytes (or other cell types), for example by in situ hybridization, and in specific cell populations for example by Northern Blot analysis and/or by in situ hybridization (Alwine et al., (1977)) and/or PCR amplification and/or LCR detection. Such nucleic acid hybridization probes have utility for in vitro screening methods for therapeutic agents (e.g., antineoplastic agents), for diagnosis and treatment of neoplastic or preneoplastic pathological conditions and genetic diseases.
A subject of the present invention is also a nucleic acid sequence which specifically hybridizes with a nucleic acid sequence of the invention as previously defined, or with their complementary sequences.
xe2x80x9cA sequence which specifically hybridizes [. . . ]xe2x80x9d is understood as meaning a sequence which hybridizes with the sequences to which it refers under the conditions of high stringency (Sambrook et al, 1989).
Such sequences can be oligonucleotides which are particularly useful as primers or probes. In that case their sequences have preferably at least 15, and more preferably at least 20 bases.
Such sequences can also be anti-sense polynucleotides. In that case their sequences have preferably the full length of the cDNA sequence coding for RB18A.
Such antisense polynucleotides are employed to inhibit transcription and/or translation of the RB1 8A mRNA species and thereby effect a reduction in the amount of the respective RB18A polypeptide in a cell (e.g., a lymphocytic leukemia cell of a patient). Such antisense polynucleotides can function as p53-modulating agents by inhibiting the formation of RB18A required for modulation of p53 function by RB18A.
In a variation of the invention, polynucleotides of the invention are employed for diagnosis or treatment of pathological conditions or genetic disease that involve neoplasia or other pathological conditions related to p53 function, and more specifically conditions and diseases that involve alterations in the structure or abundance of RB18A.
The polynucleotide sequences of the invention may be fused, by polynucleotide linkage, in frame with another polynucleotide sequence encoding a different protein (e.g., glutathione S-transferase or xcex2-galactosidase) for encoding expression of a fusion protein.
Polynucleotides of the invention can above all serve as templates for the recombinant expression of quantities of RB18A polypeptide.
A subject of the present invention is thus an isolated RB18A polypeptide substantially having the aminoacid sequence encoded by a nucleic acid sequence as above described.
More particularly the present invention relates to an isolated RB18A polypeptide or a fragment thereof comprising a sequence selected from the group consisting of a fragment from aminoacid 436 to aminoacid 1566 of SEQ ID no2, a fragment from aminoacid 436 to aminoacid 1228 of SEQ ID no2, a fragment from aminoacid 436 to aminoacid 927 of SEQ ID no2, a fragment from aminoacid 1537 to aminoacid 1566 of SEQ ID no2, a fragment from aminoacid 1234 to aminoacid 1566 of SEQ ID no2, a fragment from aminoacid 1234 to aminoacid 1406 of SEQ ID no2, a fragment from aminoacid 927 to aminoacid 1406 of SEQ ID no2, and a homologous aminoacid sequence thereof.
A preferred polypeptide of the invention is an isolated RB18A polypeptide having the aminoacid sequence of SEQ ID no2, or a homologous aminoacid sequence thereof.
xe2x80x9cA homologous aminoacid sequencexe2x80x9d is understood as meaning a sequence which differs from the sequences to which it refers by mutation, insertion, deletion or substitution of one or more aminoacids, without inducing substantial modification of biological and/or immunological properties. Said homologous aminoacid sequence shows at least 70% of homology, preferably 90% of homology with the RB18A polypeptide of SEQ ID no2.
The xe2x80x9cbiological propertiesxe2x80x9d of the polypeptides of the invention refer to the activity of the RB18A protein, such as DNA-binding, homo-oligomerization, binding to p53 and/or activation of the sequence specific DNA binding function of p53.
The xe2x80x9cimmunological propertiesxe2x80x9d of the polypeptides of the invention refer to the ability of the polypeptides of the invention to induce an immunological response mediated by antibodies, such as PAb1801 moAb, which recognize the RB18A polypeptide of the invention.
Presence on RB18A protein of common antigenic determinants with the p53 protein was demonstrated as xcex2-galactosidase-RB18A fusion proteins or in vitro translated products of RB18A cDNA were recognized by specific anti-p53 antibodies. This cross-reactivity was supported by binding on RB18A of anti-p53 moAb which reacted with 3 distinct domains of p53. Indeed, the epitopes recognized on p53 by anti-p53 moAb as DO-1, PAbl 801 and PAb421 are localized in its N or C terminal domains and are defined by aa sequence 21-25, 46-55 and 371-380, respectively (Legros et al., 1994). PAb1801, DO-1 moAb and a polyclonal anti-p53.1 Ab, prepared against the p53 N-terminal amino acid sequence 13-27, recognized the native RB18A protein. PAb421, directed against the p53 C-terminal domain, recognized only the SDS-treated form of RB18A protein. Absence of PAb421 interaction with native RB18A could be due to the masking of the PAb421 epitope in the protein core. It was also demonstrated that in vitro phosphorylation on PAb421 moAb epitope of murine (Milne et al., 1996) or human (Takenaka et al., 1995) p53 could occur in reticulocyte lysates and that these phosphorylations inhibited PAb421 moAb interaction with p53. This cross-reactivity was limited to the p53 N and C domains, as anti-p53 moAb reacting with the central domain of p53, as HO15-4, HO3-5, HO7-1, did not react with RB18A protein
However, computer analysis of amino acid sequence of the primary structure of both proteins did not allow to localize any significant identity or homology between both RB18A and p53 protein sequences. This apparent discrepancy was more likely due to the presence on RB18A of epitopes common with p53 and expressed on the tertiary structure of native RB18A protein, also partially conserved in SDS-treated RB18A protein. Similar data were obtained by Stephen et al. (1995) who reported that moAbs, including anti-p53 moAb, with known peptide reactivity, selected from 12-mer and 20-mer phage-displayed libraries not only the expected peptides but also sequences with no discernible homology with the original antigen. Presence of epitopes defined as common antigenic determinants and characterized by no primary sequence homology has been previously mentionned and called xe2x80x9cmimotopesxe2x80x9d by Geisen et al (1986, 1987). Interaction of a specific moAb with both epitope and mimotope motifs was explained by interaction with the two different ligands either of distinct paratopes on the same moAb or alternative contacts of the same paratope (Stephen et al., 1995).
In addition, RB18A protein shared some identical properties with p53 protein: indeed, RB18A protein binds to DNA, seif-oligomerize and binds to p53. This was also supported by co-immunoprecipitation of p53 and RB18A on polyclonal anti-RB18A antibody. In addition, RB18A is able to activate the sequence specific binding of p53 to DNA. All these activities, which supported the functional homology between RB18A and p53 proteins, were associated to the C-terminal domain of RB18A protein, as for p53, and more specifically to the PAb421 binding site present in this domain. Indeed: a) the domain responsible for DNA binding activity and localized on aa 927-1406, was recognized by PAb421 moAb. Part of this domain is also responsible for the self-oligomerization of RB13A (aa 1234-1406); b) in opposite to the full length RB18A, deletion of the regions recognized by PAb421 moAb abolished RB18A activation of the p53 specific DNA binding activity; c) PAb421 also defined in p53 a motif able to activate the sequence specific binding of p53 to DNA. Jayaraman and Prives (1995) showed that the C-terminus of human p53 (aa 311-393) was able to activate in trans the latent activity of sequence specific DNA binding activity of p53. A polypeptide corresponding to the p53 amino acid sequence 311 to 367 did not induce this activation suggesting that the 26 C-terminus residues were crucial for this activity. This result was confirmed by Hupp et al. (1995) who found that small peptides carrying the PAb421 epitope also stimulated the DNA-binding activity of p53.
The possibility that PAb421 moAb may define a domain which bound to DNA and/or was responsible for oligomerization has been suggested by Daud et al. (1993) and Kim et al. (1996). Indeed, PAb421 moAb cross-reacted with the mouse RAD50 protein, homologous to the yeast RAD50 protein (Daud et al., 1993), whose expression was increased in response to DNA damage. RAD50 protein carries DNA-binding oligomerization (Daud et al., 1993; Raymond and Kleckner, 1993; Kim et al., 1996), as p53. Therefore, Kim et al. (1996) suggested that the common structural epitope recognized by PAb421 Moab could imply a functional homology between RAD50 and p53. Thus, PAb421 moAb could define a motif in a protein family, including RB18A, RAD50 and p53, which carried DNA-binding and oligomerisation domains. Demonstration that a moAb may define a functionally conserved epitope on two distinct proteins has been also suggested for PAb204, an anti-T-Ag moAb. Indeed, PAb204 reacted with T-Ag and with a p68 protein (Lane and Hoeffler, 1980), both proteins exhibiting RNA helicase activity (Ford et al., 1988; Hirling et al., 1989).
Furthermore, RB18A regulated p53 specific binding on his DNA consensus binding site. Absence of detectable amount of RB18A protein in the retarded band suggested that the activation of p53 binding on DNA by RB18A was induced through an unstable (and/or low affinity) interaction between both proteins. The unstable interaction of RB18A with p53 which activated its binding to DNA was similar to the unstable interaction of p53 C-terminal domain added in trans which activated the p53 binding to his cognate DNA binding site (Jayaraman and Prives, 1995). However, a stable (and/or higher affinity) interaction between GST-RB18A and wild-type or mutated p53 was shown in absence of specific p53 DNA binding sequence. Hupp et al. (1995) suggested that interaction of the C-terminal domain of one p53 molecule with distinct region of other p53 molecule locks p53 in an inactive form for DNA binding. Addition in trans of a C-terminal peptide could promote the dissociation of the C-terminal of the full length p53 from the other region of p53. Disruption of this inhibitory interaction would induce the conformational change required to activate the DNA binding of p53. Thus, RB18A could play the same role by interacting with p53, therefore promoting conformational change of p53 and demasking its sequence-specific DNA binding domain.
Altogether, these data demonstrated that RB18A protein, which carried common antigenic and functional properties with p53, is a new protein which could regulate p53 functions.
More particularly, the RB18A protein of the invention plays an important role in the native p53 stabilization. In the absence of RB18A, native p53 has a short half-life time and consequently is hardly detected, whereas mutated p53 polypeptides are more easily detected. In the presence of RB18A, one can detect a higher level of native p53 polypeptides, due to either a stabilization of native p53 or a transformation of mutated p53 into functional p53.
Furthermore, by activating p53 and interacting with DNA non specific sequences, the RB18A polypeptide of the invention blocks the G1 phase of the cell cycle and/or regulates apoptosis. The RB18A polypeptides of the invention are thus highly interesting for controlling cell growth and apoptose, which makes them good candidates mainly for antineoplastic therapy.
On the other hand, the gene coding for the RB18A polypeptide may be induced by DNA alterations which are provoked by mutagenic agents such as UV-ray, X-ray, or chemotherapeutic agents for example. This induction would account for the failure of antineoplastic therapy using such mutagenic agents, in certain patient with a non-functional p53 (mutated and/or non-inducible p53 gene). The polypeptides or the polynucleotides of the invention could help to control this phenomenon and be used as therapeutic agents in said patients.
Besides its role in the p53 stabilization and activation, the RB18A polypeptide of the invention is also implicated in the DNA reparation by interacting with nuclear factors (e.g RXR) and helicases.
The authors of the present invention have also shown that the RB18A protein induces a reassociation of DNA double strands. In therapy, one can choose to either enhance or inhibit this renaturation. These different strategies depend on the type of cell which is targeted: tumor cells, or normal cells in a patient with cancer, after a surgery operation or after a therapy with mutagenic agents. They also depend on the type of tumor, as the cell cycle and the RB18A protein may be differently affected according to the type of tumor.
By using the FISH Mapping technique, the authors of the present invention have located the gene locus of RB18A on the chromosomal region 17q21. RB18A is thus a good candidate for preventing and/or treating diseases associated with a mutation or a loss in this region.
More particularly, as this region 17q21 also contains important genes, e.g. BRCA1, involved in the development of tumors, such as colon, breast or ovarian cancers, RB18A is expected to act as a tumor suppressor gene and to be useful for preventing and/or treating such tumors.
The polypeptides according to the invention can be obtained by any of the standard methods of purification of soluble proteins, by peptide synthesis or by genetic engineering. Said techniques comprise the insertion of a nucleic acid sequence coding for a peptide of the invention into an expression vector, such as a plasmid, and the transformation of host cells with the expression vector, by any of the methods available to the skilled person, like for instance electroporation.
The present invention thus relates to vectors for cloning and/or expression comprising a nucleic acid sequence of the invention and to host cell transfected with these vectors. The expression vector according to the invention comprises a nucleic acid sequence encoding a polypeptide of the invention, operably linked to elements allowing its expression. Such elements may be a promoter sequence, signals for initiation and termination of translation, as well as appropriate regions for regulation of translation. The insertion of said vector into the host cell may be transient or stable. Said vector may also contain specific signals for secretion of the translated protein.
These various control signals are selected according to the host cell which may be inserted into vectors which self-replicate in the selected host cell, or into vectors which integrate the genome of said host.
Host cells may be prokaryotic or eukaryotic, including but not limiting to bacteria, yeasts, insect cells, mammalian cells, including cell lines which are commercially available.
A subject of the present invention is also a method for producing a recombining RB18A polypeptide, wherein said host cell is transfected with said expression vector and is cultured in conditions allowing the expression of a polypeptide according to the invention.
Besides their use for regulating p53 protein and hence their application in the control of cell proliferation, the recombinant polypeptides of the invention are also useful for in vitro screening methods for therapeutic agents (e.g., antineoplastic agents), for diagnosis and treatment of neoplastic or preneoplastic pathological conditions and genetic diseases.
In one embodiment, candidate therapeutic agents are identified by their ability to block or increase the binding of RB18A to a p53 poiypeptide. The p53 polypeptide preferably is a full-length mature p53 protein and frequently is phosphorylated, although the phosphorylation state of individual p53 species can be variable. Typically, the p53 polypeptide comprises an amino acid sequence identical to a wild-type p53 protein sequence, although mutant p53 polypeptides are sometimes used if the mutant p53 polypeptide binds to the RB18A protein under control assay conditions (e.g., physiological conditions). Agents are tested for their ability to alter or augment binding between a p53 polypeptide and a RB18A polypeptide under suitable assay binding conditions. One means for detecting binding of a p53 polypeptide to a RB18A polypeptide is to immobilize the p53 polypeptide, such as by covalent or noncovalent chemical linkage to a solid support, and to contact the immobilized p53 polypeptide with a RB18A polypeptide that has been labelled with a detectable marker (e.g., by incorporation of radiolabelled amino acid, by epitope tagging and reporting with a fluorescent-labelled anti-epitope tag antibody, and the like). Such contacting is typically performed in aqueous conditions which permit binding of a p53 polypeptide to a RB18A polypeptide comprising a functional p53 binding site. Binding of the labelled RB18A polypeptide to the immobilized p53 is measured by determining the extent to which the labelled p53-interacting polypeptide is immobilized as a result of a specific binding interaction. Such specific binding may be reversible, or may be optionally irreversible if a cross-linking agent is added in appropriate experimental conditions. Alternatively, the p53 polypeptide may be labelled and the RB18A polypeptide immobilized. In one variation, the binding assay is performed with soluble (i.e., non-immobilized) p53 and RB18A polypeptides and the resultant bound complexes (p53:RB18A) are separated from unbound p53 and RB18A polypeptides, and the bound complexes are quantitated. Agents that inhibit or augment the formation of bound complexes as compared to a control binding reaction lacking agent are thereby identified as p53-modulating agents and are candidate therapeutic agents.
In one variation, the binding assay is performed in vivo in a cell, such as a yeast cell (e.g., Saccharomyces), and agents which inhibit or augment intermolecular complex between a p53 protein and a RB18A polypeptide are identified as p53-modulating agents. Frequently, the in vivo screening assay is a yeast two-hybrid system wherein the yeast cells express: (1) a first fusion protein comprising p53 and a first transcriptional regulatory protein sequence (e.g, GAL4 activation domain), (2) a second fusion protein comprising a RB18A polypeptide and a second transcriptional regulatory protein sequence (e.g., GAL4 DNA-binding domain), and (3) a reporter gene (e.g.,xcex2-galactosidase) which is transcribed when an intermolecular complex comprising the first fusion protein and the second fusion protein is formed. If a functional p53:RB18A complex forms, the cell expresses the reporter gene which can be detected. Agents which inhibit or augment formation of functional p53:RB18A complexes (and thus reporter gene expression) are thereby identified as p53-modulating agents.
A subject of the present invention is a composition comprising polynucleotides encoding (1) a first hybrid polypeptide comprising a p53 polypeptide and an activator domain of a transcriptional activator protein, (2) a second hybrid polypeptide comprising a RB18A polypeptide, and a DNA-binding domain of said transcriptional activator protein, and (3) a reporter polynucleotide linked to a transcriptional regulatory element whose transcriptional activity is dependent upon the presence or absence of a heterodimer comprised of the first and second hybrid polypeptide.
A further subject of the present invention is a method for identifying agents that inhibit or augment binding of a p53 polypeptide to a RB18A polypeptide to form heteromultimers, said method comprising the steps of:
performing a heterodimerization assay which includes a p53 polypeptide species comprising a binding domain with a RB18A polypeptide species comprising a binding domain and an agent under suitable binding conditions;
determining whether the agent inhibits or augments heterodimerization of the p53 polypeptide to the RB18A polypeptide;
identifying agents which inhibit or augment said heterodimerization as candidate p53 modulating agents and candidate pharmaceuticals.
A still further subject of the invention is an agent that inhibits or augments binding of a p53 polypeptide to a RB18A polypeptide as identified by the above method, as well as a pharmaceutical composition comprising such an agent in association with a pharmaceutical acceptable carrier.
The present invention also relates to monoclonal or polyclonal antibodies, or fragments thereof, or chimeric or immunoconjugate antibodies, which are capable of specifically recognizing a polypeptide according to the invention.
Polyclonal antibodies can be obtained from serum of an animal immunized against RB18A, which can be produced by genetic engineering for example, as above described, according to standard methods well-known by one skilled in the art.
Monoclonal antibodies can be obtained according to the standard method of hybridoma culture (Kohler and Milstein, 1975).
The antibodies of the present invention can be chimeric antibodies, humanized antibodies, or antigen binding fragments Fab and F(abxe2x80x2)2. They can also be immunoconjugated or labelled antibodies.
More preferred are antibodies which are directed against the C-terminal part of RB18A.
Said antibodies are particularly useful for detecting or purifying a RB18A polypeptide according to the invention in a biological sample.
Various uses of such antibodies are to diagnose and/or stage neoplasms or other cell proliferation disease states, and for therapeutic application (e.g., as cationized antibodies or by targeted liposomal delivery) to treat neoplasia, inflammation, wound healing, graft rejection, and the like.
They can be used as diagnostic reagents to identify cells exhibiting altered p53 function (e.g., preneoplastic or neoplastic cells) in a cellular sample from a patient (e.g., a lymphocyte sample, a solid tissue biopsy) as being cells which contain an increased amount of RB18A as compared to non-neoplastic cells of the same cell type(s). Additionally, anti-RB18A antibodies may be used therapeutically by targeted delivery to neoplastic cells (e.g., by cationization or by liposome/immunoliposome delivery).
Furthermore, the RB18A polypeptide is likely to interact with helicases of infectious agents such as a virus, with an affinity superior to the interaction with cellular helicases, leading to inhibition of viral infection. Consequently, the anti-RB18A antibodies of the invention could serve as an indirect marker of an undetermined viral infection.
The present invention also relates to a method for screening RB18A mutants, comprising the steps of:
providing RB18A derivative polypeptides, wherein a RB18A xe2x80x9cderivative polypeptidexe2x80x9d means a polypeptide which differs from an isolated RB18A polypeptide of the invention by mutation, insertion, deletion or substitution of one or more aminoacids, without inducing substantial modification of biological and immunological properties;
testing said RB18A polypeptides for their binding affinity to native p53 protein and/or helicases of infectious agents;
identifying RB18A derivative polypeptides which bind to native p53 protein and/or helicases of infectious agents with an affinity superior to the isolated RB18A polypeptide of the invention, as RB18A mutants of interest.
Such RB18A mutants are good candidate for preventing and/or treating a disease involving p53 and/or an infection.
The present invention also encompasses a pharmaceutical composition comprising a RB18A mutant identified by the above method, in association with a pharmaceutically acceptable carrier.
Another subject of the present invention is a pharmaceutical composition comprising a purified RB18A polypeptide of the invention and/or a homologous polypeptide thereof, or an isolated nucleic acid sequence encoding said polypeptides in association with a pharmaceutically acceptable carrier.
The nucleic acid sequences of the invention may be administered in a naked form or in association with transfection-facilitating agents. They are preferably inserted in an appropriate vector to facilitate its penetration into the cell according to standard methods well-known by those skilled in the art.
In a gene therapy strategy, they may be more particularly inserted in retrovirus in association with a promoter specific of tumor cells, leading to the apoptosis of tumor cells.
A further subject of the present invention is a pharmaceutical composition comprising an anti-sense sequence capable of specifically hybridizing with a nucleic acid sequence encoding said polypeptides, in association with a pharmaceutically acceptable carrier.
A still further subject of the present invention is a pharmaceutical composition comprising an antibody specifically directed against said polypeptides, in association with a pharmaceutically acceptable carrier.
The pharmaceutical compositions of the invention which comprise a purified RB18A polypeptide or an isolated nucleic acid sequence encoding said polypeptide are useful for preventing or treating a variety of human and veterinary diseases, such as neoplasia, inflammation, wound healing, graft rejection reperfusion injury, myocardial infarction, stroke, traumatic brain injury, neurodegenerative diseases, aging, ischemia, toxemia, infection, AIDS and hepatitis. Yet, they are preferably used as antineoplastic compositions, or as compositions directed against any other cell proliferation disease. They can be in particular advantageously used in the treatment of the colon, breast of ovarian cancer.
The pharmaceutical compositions of the invention may be administered to a mammal, preferably to a human, in need of a such treatment, according to a dosage which may vary widely as a function of the age, weight and state of health of the patient, the nature and severity of the complaint and the route of administration.
The appropriate unit forms of administration comprise oral forms such as tablets, gelatin capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, subcutaneous, intramuscular, intravenous, intranasal or intraoccular administration forms and rectal administration forms.
A further subject of the present invention is a method of preventing and/or treating a disease involving the RB18A protein, such as neoplasms or other cell proliferation disease, which comprises administering to a subject in need of a such treatment an amount of a pharmaceutical composition as above defined effective to prevent and/or alleviate said disease.
The present invention is further illustrated by, but not limited to, the figures and the examples that follow.